Isolation, diversity and antimicrobial activity of sponge-associated bacteria from South Australian marine environments

Author: Yitayal Anteneh

Anteneh, Yitayal, 2020 Isolation, diversity and antimicrobial activity of sponge-associated bacteria from South Australian marine environments, Flinders University, College of Medicine and Public Health

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The world is in the search of novel antibiotics from various sources as current epidemics due to multidrug-resistant microbes threaten the existence of life. Since the terrestrial environments have been exploited for many years for the search of compounds, the probability of getting new active compounds from these sources is low. Therefore, considering less explored sources is essential and marine sponges have received some attention as novel biologically active compounds are increasingly reported from them. Most studies claimed that many of the current discovered active metabolites from marine sponges originated from associated symbionts rather than the host sponges. As a result, it is important to study the diversity of microorganisms from sponges from various sources. This study was designed to isolate bacteria from marine sponges of South Australia to investigate their diversity, factors affecting diversity and antimicrobial activities of the isolates against human microbial pathogens. Previous molecular-based studies in this area indicated more than 90% of the microbial community in the sponges are unculturable. This study attempted to culture this bacterial population by employing multiple cultivation strategies.

Twelve sponge samples were collected from Rapid Bay and Glenelg beaches of South Australia at a depth of 10-15 m. The bacterial population was isolated using seven primary isolation agar media which were incubated at different temperatures and oxygen levels over 3 months. Bacterial isolates were identified and categorized with the help of microscopic and macroscopic features, and restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA gene PCR products. Subsequently, selection of bacterial isolates were identified using their 16S rRNA gene sequence. The bacterial isolates were tested for antimicrobial activities against human pathogenic bacteria and fungi. For selected active strain, antimicrobial produced in large scale, purified and identified.

A total of 1234 bacterial colonies were isolated, and these were categorised into 383 types based on micro and macroscopic features. Enzymatic digestion of 16S rRNA gene PCR products produced 38 RFLP patterns. 16S rRNA gene sequencing of representatives from each pattern identified four phyla; Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, containing 21 genera. Actinobacteria was the most dominant phylum isolated, while Streptomyces was the most frequent genus isolated. Three novel bacterial isolates and one novel fungus were identified using polyphasic taxonomy Comparison of culture-dependent and independent approaches revealed discrepancies, where genera observed by isolation not seen by next generation sequencing.

A significant variation of bacterial diversity was observed depending on the growth media, sponge collection sites, sponge types, time of incubation and incubation conditions. Furthermore, the bacterial isolates markedly varied depending on their temperature tolerance and NaCl preference for growth.

A total of 169 bacteria were tested for antimicrobial activities against human pathogenic bacteria and fungi. Approximately 41% of the tested bacterial strains displayed antimicrobial activities. Specifically, 37% of the bacterial isolates showed activity against multidrug-resistant Staphylococcus aureus, and 21% of them produced an antifungal activity at least against one of the tested fungi. Antimicrobial production was highly influenced by media type and incubation period. A novel water soluble antibiotic effective against multidrug-resistant Staphylococcus aureus was discovered.

The findings of the study indicated that the marine sponges of South Australia are the sources of novel and highly diverse bacterial strains. The isolation approaches helped to report 5 genera which were not reported in previous related studies. Furthermore, the approaches resulted in the isolation of many uncommon genera at a time compared to similar studies. The success of isolation of highly diverse and novel bacterial isolates depends on various factors and considering them during future studies helps to reflect the true picture of the culturable bacterial population from sponges.

Antimicrobial screening and further analysis indicated that the bacterial isolates from these sites could be alternative sources for the search of new antimicrobials effective against multidrug-resistant microorganisms. Other colleagues in the research group also reported herbicidal and anticancer activities from these bacterial strains. They also produced biologically active enzymes such as alginate lyase. These diverse biological activities may stimulate many scholars to exploit the area for the search of different biological applications of sponge-associated bacteria.

Keywords: Marine sponges, Bacteria, Actinobacteria, Antimicrobial, Australia

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Medicine and Public Health
Supervisor: Prof.Chris Franco